5 key misunderstandings in the development and production of chemiluminescence!
Chemiluminescence immunoassay (CLIA) combines highly sensitive chemiluminescence technology with highly specific immune response to establish a chemiluminescence immunoassay. CLIA has the characteristics of high sensitivity, strong specificity, wide linear range, easy operation, and low cost of use. CLIA has a wide range of applications, not only for the detection of antigens, haptens and antibodies of different molecular sizes, but also for the detection of nucleic acid probes. Due to its advantages, the number of in vitro diagnostic tests based on this technology is increasing day by day. And whenever a chemiluminescence analyzer is installed in a hospital or a diagnostic laboratory, the demand for its corresponding kits may reach thousands of sets a day.
Therefore, the demand for CLIA IVD kits has begun to soar, but the early small-scale production, pure manual operation and simple quality control methods can no longer meet the needs of large-scale production. How to ensure the consistency between batches; how to ensure the stability of quality after scale-up of production; how to ensure the effectiveness of the final application of the finished product (finally using no more than a few hundred microliters in the analyzer) are all produced as CLIA IVD kits key. During more than a decade of development, we have identified five key pitfalls in the biomagnetic separation process. These misunderstandings are easy to ignore but often delay the project schedule, cause significant economic losses, and sometimes even put production at risk. Therefore, correctly mastering the technical information of magnetic separation is the key to ensure the success of product development and production.
How to describe a biomagnetic separation process?
When a new CLIA-IVD kit is transferred from the R&D phase to the production phase, all production operating parameters need to be readjusted to accommodate the new throughput and processing volume. The specifications of biomarkers, buffers and coating operations all benefit from the experience accumulated in the production of non-magnetic kits. The coupling of antibodies and magnetic beads is very similar to colloidal gold or latex particles, but the operation of the washing process using magnetic separation is very different from the production of other non-CLIA-IVD kits.
Although the use of biomagnetic separation in the separation phase of manufacture of CLIA kits seems to be the obvious choice, there are still some problems in practice. The first is to describe the entire process itself. When communicating with IVD kit manufacturers, they often mentioned the following aspects in terms of biomagnetic separation:
Separation time: the time to separate the solid phase from the buffer system.
Bead Loss: The maximum amount of beads (and conjugated biomarkers) lost during production.
Batch processing volume: the batch size to be processed, and even the processing volume is elastic in some cases. To avoid irreversible aggregation: If irreversible aggregation of magnetic beads occurs during the production process, it needs to be resuspended in various ways. After suspension, it must be checked whether it is handled properly. Because each kit (ml level) requires the same properties, incorrect resuspension handling can increase batch-to-batch variability.
However, these are "functional" parameters, they are the result of magnetic separation rather than factors affecting the separation process. In the whole process of biomagnetic separation, what is the parameter that is really missing but defines the whole separation process?
A key parameter in the biomagnetic separation process is magnetic force. Magnetic beads move at a specific velocity, a net force due to the competition between magnetic force and drag force, the latter caused by buffer viscosity
Misunderstanding 1: It is always due to bad magnetic beads
When choosing magnetic beads, users are very concerned about whether they have selected the "correct" magnetic beads. Assuming that the appropriate biomarker and the perfect coupling/coating method have been selected, then the "correct" magnetic beads selected by the user should have the following characteristics:
1) High recovery/rapid separation, matched to time in analytical equipment, magnetic separation fast enough to scale up without loss of large volumes of magnetic beads and coupled biomarkers?
2) No aggregation problem, magnetic beads can be easily remixed and suspended. Even if bead aggregation can be restored with a few additional steps of sonication? (but this is difficult to control and manipulate during mass production)
3) Low batch-to-batch variation, each batch aliquot (usually less than milliliters) and production batch (upgrade) must be consistent. If not, the difference affects the results given by the analyzer
What happens if the above requirements are not met?
The most common response from kit manufacturers to defective magnetic separation results is that the choice of magnetic beads is "wrong". Then the manufacturer contacts the magnetic bead supplier (or an alternative supplier) for a long-term discussion on the magnetic beads, re-examines the coating method, etc. If it falls into this stage, the development and launch of new products will be seriously affected, and the progress will be greatly delayed.
TIP: If the biomarkers are well coupled to the beads, changing the beads if there is a problem will be an expensive and time consuming exercise, while still not effectively solving the problem!
Manufacturers should get rid of the entanglement of "correct" and "wrong" magnetic beads, and focus on biomagnetic separation equipment and equipment. By comparing the following figures, we can find that the same suspended magnetic beads will behave completely differently under the action of different biomagnetic separators
Figure 2 shows the slow separation of magnetic beads handled by a traditional magnetic stand (blue) on the left.
This means longer time to complete the separation or higher loss of beads and biomarkers. However, if one waits for a longer separation time to avoid loss, some beads will suffer from irreversible aggregation problems, as the bead that separates first (closest to the vial wall) is subjected to very strong magnetic forces squeezing the beads in the Together. If in this separation step, we do not consider the influence of the separation equipment, but only judge from the separation performance of the magnetic beads, then we will get a wrong conclusion: we must replace other magnetic beads to solve the problem. At the same time, we can observe from Figure 2 that the same magnetic bead suspension passes through the advanced biomagnetic separation device (orange on the right) and we can see that the separation is completely different. The magnetic beads are quickly separated at the same speed, and due to the uniform magnetic force, the wall-attached magnetic beads are subjected to mild magnetic force in a shorter time, eliminating the risk of irreversible aggregation.
